A biomolecule may be defined as any molecule or substance of biological interest. The biomolecule may have its own biological activity or simply be engaged in a biological reaction. The biomolecule can be a ligand, binder, protein, enzyme, antigen, antibody, nucleic acid, vitamin, dye, substrate, cofactor, etc. Immobilized biomolecules have been widely used for a variety of applications including bioaffinity separations, immunoassays, bioreactors and biosensors. One of the most difficult problems in immobilizing a biomolecule to a solid or liquid carrier is achieving the efficiency and stability of attachment. In an attempt to overcome this difficulty, the covalent attachment technique, involving derivatization of the carrier surface to chemically couple with a biomolecule, has been commonly used. The biomolecule can be immobilized directly or immobilized via a linker. However, this approach has well known disadvantages including introduction of reactive groups to the carrier surface often leading to increased nonspecific binding, significant loss of reactivity of the immobilized biomolecule, and permanent attachment of the biomolecule preventing it from being recovered for re-use. Recoverability is an important aspect of immobilization when the biomolecule is a scarce or very expensive reagent.
The general covalent attachment methods will not be reviewed in this application but those employing a perfluorocarbon polymer-based carrier will be addressed. Halbert et al., U.S. Pat. No. 4,642,285, issued Feb. 10, 1987, disclose a method of covalently attaching proteins such as an antibody onto an insoluble member. To effect this bonding, the insoluble member used must be provided with reactive groups or sites capable of reacting with the specific antibody used. The insoluble member disclosed in this reference is a commercially available material known as PROTAPOL DI/1 from Imperial Chemical Industries of Australia and New Zealand (ICIANZ). The material is available in a disc form and comprises a polytetrafluoroethylene (PTFE) backbone having isothiocyanopolystyrene groups grafted uniformly over its surface. The method provides a means for directly immobilizing macromolecules via the reactive groups introduced to the chemically inert perfluorocarbon solid carrier. However, the method does not take advantage of the inherent low nonspecific binding properties of the PTFE polymer and suffers from the known disadvantages described above.
Tregear et al., U.S. Pat. No. 3,700,609, issued Oct. 24, 1972, also disclose a graft copolymer such as those described by Halbert et al. The copolymer comprises a polymeric backbone, for example, PTFE, onto which is grafted a different copolymerizable comonomer having substituent groups capable of forming a chemical bond with a protein. A preferred protein reactive group is the isothiocyanate group or the chloromethyl group. Again, the method is for a direct covalent attachment of biomolecules onto the surface of the copolymer having reactive groups.
Sarfaty et al., U.S. Pat. No. 3,639,516, issued Feb. 1, 1972, also disclose a graft copolymer comprising a polymeric nucleus such as PTFE or polytrifluoromonochloro ethylene polymer, and surface grafted on to it copolymeric side chains comprising a copolymer such as styrene or substituted styrene. The difference is that the graft copolymer in this patent is designed to have specific reactivities toward carbonylic steroids. The invention describes the use of such a copolymer for separation of carbonylic steroids.
Yen et al., U.S. Pat. No. 4,035,316, issued Jul. 12, 1977, describe a method for isolation of specific cells by density gradient centrifugation. The technique involves labeling of cells to be separated with lectin or cell-specific antibody attached to microspheres. The microspheres comprise a biocompatible copolymer, for example, a hydroxy or amine substituted acrylic monomer such as hydroxyethyl methacrylate with a light or dense comonomer such as a fluoromonomer. Here, the fluoromonomer such as trifluoroethyl methacrylate, hexafluoroisopropyl methacrylate or pentafluorostyrene in the generation of microspheres is used to provide a density adequate for separation of the labeled cells by sedimentation or centrifugation techniques. The same method is also disclosed in a Divisional of the above patent, U.S. Pat. No. 4,105,598, issued Aug. 8, 1978.
Biebricher et al., U.S. Pat. No. 4,177,038, issued Dec. 4, 1979, describe a method to permit biologically active matter to be bonded to an insoluble vehicle in such a manner that the capability of the biological substance of interacting with another biological substance is not impaired. The method uses polyethylene glycol as a linker to extend the distance between a biological material and the carrier. Similarly, Davis et al., U.S. Pat. No. 4,179,337, issued Dec. 18, 1989, disclose a method of coupling polypeptides to polyethylene glycol or polypropylene glycol to provide physiologically active non-immunogenic water soluble compositions for injection.
In general, the use of polyglycols imparts protection to sensitive biologically active molecules but fails to provide an adequate number of functional groups to which to attach other chemical entities.
Another commonly used method of immobilization is a simple adsorption technique. The major disadvantage of this approach is the weakness of the attachment. Adsorption involves complex nonspecific hydrophobic, charge and Van der Waals interactions between the biomolecule, reaction medium and surface of the carrier. Thus, the efficiency of attachment or reactivity of the immobilized biomolecule is rather unpredictable. Furthermore, due to the nonspecific nature of interactions, the problem of nonspecific binding is apparent. The immobilized biomolecule often leaches off during use and is sensitive to reaction conditions such as pH, ionic strength and buffer type.
A novel method for noncovalent attachment of an affinity ligand or binder onto a solid or liquid perfluorocarbon carrier has been disclosed in pending applications, U.S. Ser. Nos. 07/032,642, filed Mar. 31, 1987, now abandoned; 07/134,026, filed Dec. 17, 1987, now abandoned; 07/020,808, filed Mar. 2, 1987, now U.S. Pat. No. 4,885,250, issued Dec. 5, 1989, and 07/134,028, filed Dec. 17, 1987, now U.S. Pat. No. 4,954,444, issued Sep. 4, 1990. U.S. Ser. No. 07/032,642 is a continuation-in-part of 06/863,607, filed May 15, 1986; both have since been abandoned. The immobilization technique employs chemical modification of a ligand or binder to effect a specific interaction between the ligand or binder with the carrier. The ligand or binder is modified to introduce a perfluoroalkyl group which anchors onto the perfluorocarbon surface of the carrier by the strong fluorophilic interactions. The invention in these applications offers a way to securely but reversibly immobilize the ligand or binder onto a chemically inert surface having low nonspecific binding properties. While the method is widely applicable for preparation of affinity supports or immobilized enzymes, it is most effective with the ligand or binder having multiple reactive groups for substitution with multiple perfluoalkyl anchoring groups.
A small ligand such as a hapten or a dye molecule often has only one reactive group. Perfluoroalkylation of such ligands will anchor onto a perfluorocarbon surface but the attachment is not as secure, thus resulting in potential desorption of the ligands upon change in pH or ionic strength. It is an object of this invention to provide an improved means especially useful for securely attaching small biomolecules. In the case of enzymes, perfluoroalkylation at multiple sites may lead to a reduction in specific activity. Furthermore, immobilization onto a carrier through the multiple anchoring groups may enhance unfolding of the protein resulting in a severe reduction in specific activity. It is another object of this invention to provide an improved means useful for securely attaching enzymes or other proteins having biological activity without compromising their reactivity.
Smith et al., U.S. Pat. No. 4,267,273, issued May 12, 1981, disclose a method for recovering enzymes from the reaction medium for re-use. To accomplish the separation, an enzyme is chemically modified to attach sufficient non-polar groups, such that on contacting the preparation in aqueous media with an inert water-immiscible liquid, the preparation becomes associated with said water-immiscible liquid separated from the aqueous medium. Sometimes, it is not possible to attach non-polar groups directly to the enzyme without significantly reducing its activity. Thus, it is often preferable to prepare the enzyme bonded to or adsorbed on a polymeric support, which may then carry multiple non-polar groups. The method is an improvement over the conventional use of immobilized enzymes on an insoluble solid support or of water-soluble enzyme-polymer complexes obviating the need to filter or centrifuge for recovery of the enzyme. However, the invention does not teach how to improve attachment of a protein such as an enzyme to the surface of a carrier.
A publication by De Miguel et al., Chromatographia, Vol 24, 849-853, 1987, reports that the specific retention of perfluorinated(PF) compounds on PF stationary phases grows exponentially by increasing the chain length and the number of chains within the same compound. A strong cooperative effect is observed with the multi-strand compounds. For example, the double strand bis 1,2 (heptafluorobutanoyl-oxyl) butane having two three-carbon backbone perfluoro groups is more retained than the single strand (tridecafluoroheptanoyloxyl) butane although the number of fluorinated carbon atoms is the same. Based on these observations, the authors speculate that the tremendous retention power introduced by these multi-strand compounds should allow the dynamic anchoring of biomolecules. However, this aspect of the invention has been fully demonstrated previously by the disclosures in pending applications referred to above. In fact, a recognition that small molecules having only one reactive group would not result in a secure attachment has led to an improved method disclosed in the present application.
It is clear that currently available immobilization methods suffer from various disadvantages. The present invention provides an improved method especially useful for securely but reversibly anchoring small biomolecules having only one reactive group as well as enzymes or other proteins without compromising their reactivity onto a perfluorocarbon polymer-based carrier.